Complete genome sequencing and investigation on the fiber-degrading potential of Bacillus amyloliquefaciens strain TL106 from the tibetan pig.

BACKGROUND: Cellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens, TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro. RESULTS: TL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including ß-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and ß-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and ß-glucan of wheat and highland barley . CONCLUSIONS: The genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and ß-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and ß-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.

Multi-copy expression of a protease-resistant xylanase with high xylan degradation ability and its application in broilers fed wheat-based diets.

The acidic thermostable xylanase (AT-xynA) has great potential in the feed industry, but its low activity is not conductive to large-scale production, and its application in poultry diets still needs to be further evaluated. In Experiment1, AT-xynA activity increased 3.10 times by constructing multi-copy strains, and the highest activity reached 10,018.29 ± 91.18 U/mL. AT-xynA showed protease resistance, high specificity for xylan substrates, xylobiose and xylotriose were the main hydrolysates. In Experiment2, 192 broilers were assigned into 3 treatments including a wheat-based diet, and the diets supplemented with AT-xynA during the entire period (XY-42) or exclusively during the early stage (XY-21). AT-xynA improved growth performance, while the performance of XY-21 and XY-42 was identical. To further clarify the mechanism underlying the particular effectiveness of AT-xynA during the early stage, 128 broilers were allotted into 2 treatments including a wheat-based diet and the diet supplemented with AT-xynA for 42 d in Experiment3. AT-xynA improved intestinal digestive function and microbiota composition, the benefits were stronger in younger broilers than older ones. Overall, the activity of AT-xynA exhibiting protease resistance and high xylan degradation ability increased by constructing multi-copy strains, and AT-xynA was particularly effective in improving broiler performance during the early stage.

Effect of dietary supplementation with recombinant human lysozyme on growth performance, antioxidative characteristics, and intestinal health in broiler chickens.

Lysozyme is often used as a feed additive to act as an antibacterial protein that boosts the immune system of livestock and poultry while protecting against pathogens. To investigate the effects of recombinant human lysozyme (rhLYZ) from Pichia pastoris and chlortetracycline on broiler chicken's production performance, antioxidant characteristics, and intestinal microbiota, a total of 200, 1-d-old male Arbor Acres broiler chickens (46.53 ±â€…0.42 g) were selected for a 42-d experiment. Dietary treatments included a basal diet of corn-soybean meal supplemented with either 0 mg/kg (CON), 50 mg/kg aureomycin (ANT), 20 mg/kg rhLYZ (LOW), 60 mg/kg rhLYZ (MEDIUM), or 180 mg/kg rhLYZ (HIGH). Compared with CON, MEDIUM diet increased (P < 0.05) average daily gain (67.40 g) of broilers from day 22 to 42. In the early (1.29) and overall phases (1.69), MEDIUM led to a reduction (P < 0.05) in the feed conversion ratio of broiler chickens. Furthermore, in comparison to the CON and ANT, MEDIUM exhibited reduced (P < 0.05) levels of INF-γ and tumor necrosis factor-α in the serum. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was lower (P < 0.05) in the MEDIUM treatment compared to CON. Overall, supplementation of 60 mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function, while also influencing the composition of intestinal microbiota. This suggests lysozyme's potential to replace antibiotic additives in feed.

The aim of this study was to explore the effects of recombinant human lysozyme (rhLYZ) produced from Pichia pastoris and chlortetracycline on broiler chicken performance, antioxidant properties, and gut microbiota. A 42-d experiment was conducted, involving 200 1-d-old male Arbor Acres broiler chickens. We provided different diets: a standard diet (CON), a diet with 50 mg/kg aureomycin (ANT), a diet with 20 mg/kg rhLYZ (LOW), a diet with 60 mg/kg rhLYZ (MEDIUM), or a diet with 180 mg/kg rhLYZ (HIGH). The results showed that, compared to the control group, the MEDIUM group significantly increased the average daily gain of broilers to 67.40 g from day 22 to 42. Additionally, the MEDIUM group exhibited a reduced feed conversion ratio during both the early and overall growth stages of the chickens. Furthermore, serum levels of INF-γ and tumor necrosis factor-α were lower in the MEDIUM group compared to both the CON and ANT groups. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was also lower in the MEDIUM treatment compared to the CON group. Overall, supplementation with 60 mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function in broiler chickens while also influencing the composition of their intestinal microbiota. This suggests the potential of lysozyme as a replacement for antibiotic additives in feed.

Expression and Characterization of Laccase Lac1 from Coriolopsis trogii Strain Mafic-2001 in Pichia pastoris and Its Degradation of Lignin.

The laccase gene (Lac1) was cloned from Coriolopsis trogii strain Mafic-2001. Full-length sequence of Lac1 containing 11 exons and 10 introns is composed of 2140 nucleotides (nts). mRNA of Lac1 encoded for a protein of 517 aa. Nucleotide sequence of the laccase was optimized and expressed in Pichia pastoris X-33. SDS-PAGE analysis showed that the molecular weight of the purified recombinant laccase rLac1 was about 70 kDa. The optimum temperature and pH of rLac1 were 40 ℃ and 3.0, respectively. rLac1 showed high residual activity (90%) in the solutions after 1 h incubation at the pH ranging from 2.5 to 8.0. rLac1 maintained over 60% of laccase activity at the temperatures ranging from 20 to 60 °C, and kept higher than 50% of its activity at 40 °C for 2 h. The activity of rLac1 was promoted by Cu2+ and inhibited by Fe2+. Under optimal conditions, lignin degradation rates of rLac1 on the substrates of rice straw, corn stover, and palm kernel cake were 50.24%, 55.49%, and 24.43% (the lignin contents of substrates untreated with rLac1 were 100%), respectively. Treated with rLac1, the structures of agricultural residues (rice straw, corn stover, and palm kernel cake) were obviously loosened which was reflected by the analysis of scanning electron microscopy and Fourier transform infrared spectroscopy. Based on the specific activity of rLac1 on the degradation of lignin, rLac1 from Coriolopsis trogii strain Mafic-2001 has the potential for in-depth utilization of agricultural residues.

The Effects of Dietary Bacillus amyloliquefaciens TL106 Supplementation, as an Alternative to Antibiotics, on Growth Performance, Intestinal Immunity, Epithelial Barrier Integrity, and Intestinal Microbiota in Broilers.

A total of 240 1-day-old Arbor Acres male broilers were randomly divided into five dietary treatments (control feed (CON), supplemented with 75 mg/kg aureomycin (ANT), supplemented with 7.5 × 108 CFU/kg (Ba1) and 2.5 × 109 CFU/kg (Ba1), and 7.5 × 109 CFU/kg (Ba3) Bacillus amyloliquefaciens TL106, respectively) to investigate the probiotic effect of TL106 instead of antibiotics in broilers. On days 1−21, the average daily gain of broilers in the Ba groups was increased compared with the CON group (p < 0.05). In addition, the feed/gain ratio of broilers in the Ba groups was lower than that of broilers in the CON and ANT groups on days 22−42 and days 1−42 (p < 0.05). Compared with the CON group, dietary TL106 increased the digestibility of crude fiber and crude protein (p < 0.05), and the effect was similar to that of the ANT group. The levels of IL-1ß, IFN-γ, and IL-6 in serum, jejunum, and ileum of broilers fed TL106 were decreased compared with the control group (p < 0.05). The mRNA expression of tight junction proteins in broilers of ANT and Ba groups was higher than the control group (p < 0.05). After 21 days, villus height and the ratio of villus height to crypt depth of duodenum and jejunum of broilers fed TL106 were higher than the control group (p < 0.05). The concentrations of short-chain fatty acids such as lactate, acetate, propionate, and butyrate in cecal digesta of broilers dietary TL106 were higher than the control group (p < 0.05). The supplementation with TL106 altered the compositions and diversity of the cecal microbiota of broilers. Moreover, supplementation with TL106 improved the ratio of Firmicutes to Bacteroidetes and decreased the relative abundance of Proteobacteria on days 21 and 28, while the abundance of Peptostreptococcaceae, Ruminococcaceae and Lactobacillaceae was increased. On days 35 and 42, broilers fed TL106 had an increased total abundance of Firmicutes and Bacteroidetes and decreased abundances of Lactobacillaceae, while the abundance of Barnesiellaceae was increased. In conclusion, dietary supplementation with TL106 improved the broiler's growth performance, immune response capacity, gut health, modulated development, and composition of the gut microbiota in broilers. It is suggested that Bacillus amyloliquefaciens TL106 may be a suitable alternative to in-feed antibiotics to improve broiler health and performance.

Characterization of a novel GH10 xylanase with a carbohydrate binding module from Aspergillus sulphureus and its synergistic hydrolysis activity with cellulase.

A study was carried out to investigate the characterization of a novel Aspergillus sulphureus JCM01963 xylanase (AS-xyn10A) with a carbohydrate binding module (CBM) and its application in degrading alkali pretreated corncob, rapeseed meal and corn stover alone and in combination with a commercial cellulase. In this study, the 3D structure of AS-xyn10A, which contained a CBM at C-terminal. AS-xyn10A and its CBM-truncated variant (AS-xyn10A-dC) was codon-optimized and over-expressed in Komagaella phaffii X-33 (syn. Pichia pastoris) and characterized with optimal condition at 70 °C and pH 5.0, respectively. AS-xyn10A displayed high activity to xylan extracted from corn stover, corncob, and rapeseed meal. The concentration of hydrolyzed xylo-oligosaccharides (XOSs) reached 1592.26 µg/mL, 1149.92 µg/mL, and 621.86 µg/mL, respectively. Xylobiose was the main product (~70%) in the hydrolysis mixture. AS-xyn10A significantly synergized with cellulase to improve the hydrolysis efficiency of corn stover, corncob, and rapeseed meal to glucose. The degree of synergy (DS) was 1.32, 1.31, and 1.30, respectively. Simultaneously, XOSs hydrolyzed with AS-xyn10A and cellulase was improved by 46.48%, 66.13% and 141.45%, respectively. In addition, CBM variant decreased the yields of xylo-oligosaccharide and glucose in rapeseed meal degradation. This study provided a novel GH10 endo-xylanase, which has potential applications in hydrolysis of biomass.

Effects of Xylanase in Corn- or Wheat-Based Diets on Cecal Microbiota of Broilers.

Xylanase has been demonstrated to improve growth performance of broilers fed wheat- or corn-based diets due to its ability to degrade arabinoxylans (AX). However, content and structure of AX in corn and wheat are different, comparing effects of xylanase on cecal microbiota of broilers fed corn- or wheat-based diets could further elaborate the mechanism of the specificity of xylanase for different cereal grains. Thus, a total of 192 one-day-old broilers were randomly allotted into four dietary treatments, including wheat-soybean basal diet, wheat-soybean basal diet with 4,000U/kg xylanase, corn-soybean basal diet, and corn-soybean basal diet with 4,000U/kg xylanase to evaluate interactive effects of xylanase in corn- or wheat-based diets on broilers cecal microbiota during a 6-week production period. The results indicated that bacterial community clustering was mainly due to cereal grains rather than xylanase supplementation. Compared with broilers fed wheat-based diets, corn-based diets increased alpha-diversity and separated from wheat-based diets (p<0.05). Xylanase modulated the abundance of specific bacteria without changing overall microbial structure. In broilers fed wheat-based diets, xylanase increased the abundance of Lactobacillus, Bifidobacterium, and some butyrate-producing bacteria, and decreased the abundance of non-starch polysaccharides-degrading (NSP) bacteria, such as Ruminococcaceae and Bacteroidetes (p<0.05). In broilers fed corn-based diets, xylanase decreased the abundance of harmful bacteria (such as genus Faecalitalea and Escherichia-Shigella) and promoted the abundance of beneficial bacteria (such as Anaerofustis and Lachnospiraceae_UCG_010) in the cecum (p<0.05). Overall, xylanase supplementation to wheat- or corn-based diets improved broilers performance and cecal microbiota composition. Xylanase supplementation to wheat-based diets increased the abundance of butyrate-producing bacteria and decreased the abundance of NSP-degrading bacteria. Moreover, positive effects of xylanase on cecal microbiota of broilers fed corn-based diets were mostly related to the inhibition of potentially pathogenic bacteria, and xylanase supplementation to corn-based diets slightly affected the abundance of butyrate-producing bacteria and NSP-degrading bacterium, the difference might be related to lower content of AX in corn compared to wheat.

Administration of Saccharomyces boulardii mafic-1701 improves feed conversion ratio, promotes antioxidant capacity, alleviates intestinal inflammation and modulates gut microbiota in weaned piglets.

BACKGROUND: Probiotics are used as a means to improve animal health and intestinal development. Saccharomyces boulardii is a well-known probiotic; however, few studies have examined the effects of S. boulardii on weaned piglet performance. Therefore, this 28-day study compared the effects of S. boulardii mafic-1701 and aureomycin in diets for weaned piglets on growth performance, antioxidant parameters, inflammation and intestinal microbiota. One hundred and eight piglets, weaned at 28 d of age (8.5 ± 1.1 kg), were randomly divided into the three dietary treatment groups with six pens and six piglets per pen (half male and half female). The dietary treatment groups were as follows: 1) basal diet (CON); 2) basal diet supplemented with 75 mg/kg aureomycin (ANT); 3) basal diet supplemented with 1 × 108 CFU/kg S. boulardii mafic-1701 (SB). RESULTS: Compared to CON group, SB group had higher feed efficiency (P < 0.05) in the last 14 d and lower diarrhea rate (P <  0.05) over the entire 28 d. Total superoxide dismutase in serum was markedly increased in SB group (P < 0.05). Moreover, compared with CON group, SB group decreased the levels of pro-inflammatory cytokines interleukin-6 (P <  0.01) and Tumor necrosis factor-α (P < 0.05) in jejunum. Supplementation of S. boulardii mafic-1701 increased the abundance of Ruminococcaceae_UCG_009 and Turicibacter (P < 0.05), whereas the abundance of unclassified_Clostridiaceae_4 was decreased (P < 0.05). Furthermore, S. boulardii mafic-1701 administration increased cecal concentration of microbial metabolites, isobutyrate and valerate (P < 0.05). CONCLUSIONS: The improvement in feed conversion ratio, reduction in diarrhea rate in weaned piglets provided diets supplemented with S. boulardii mafic-1701 may be associated with enhanced antioxidant activity, anti-inflammatory responses and improved intestinal microbial ecology.

High-level expression of an acidic thermostable xylanase in Pichia pastoris and its application in weaned piglets.

An acidic thermostable xylanase (AT-xynA) which was stable at low pH and high temperature was considered to have great potential in animal feed. For large-scale production, AT-xynA activity was enhanced about 1-fold in Pichia pastoris by constructing a double-copy expression strain in this study. Furthermore, impacts of different AT-xynA levels on growth performance, nutrient digestibility, short-chain fatty acids, and bacterial community in weaned piglets were determined. Compared with the control group, ADFI and ADG were higher for the pigs fed 4,000 or 6,000 U/kg AT-xynA (P < 0.05). AT-xynA supplementation also significantly increased the digestibility of OM, GE, and DM (P < 0.05). AT-xynA supplementation increased the concentrations of acetate in ileal (P < 0.01) and cecal digesta (P < 0.05). Isobutyrate (P < 0.05) and valerate (P < 0.05) concentrations in colonic digesta also significantly increased compared with the control group. AT-xynA supplementation increased the abundance of Lactobacillus in the ileal, cecal, and colonic digesta of weaned piglets (P < 0.05). AT-xynA alleviated anti-nutritional effects of nonstarch polysaccharides (NSP) by preventing the growth of Pateurella and Leptotrichia in the ileum (P < 0.05). AT-xynA increased the abundance of NSP-degrading bacteria, such as Ruminococcaceae, Prevotella in the cecum and colon (P < 0.05). In summary, AT-xynA addition could improve the growth performance of weaned piglets by altering gut microbiota.